The long-term objective of this project is to understand the molecular pathology and pathogenesis of X-linked adrenoleukodystrophy (ALD), a fatal neurodegenerative disorder. Defective peroxisomal activation of very long-chain fatty acids (VLCFA) by very long-chain fatty acyl-CoA synthetase (VLCS) causes elevated VLCFA levels in ALD patients. The product of the gene shown to be defective in ALD (ALDP) is a peroxisomal membrane protein that is a member of the ATP-binding cassette (ABC) membrane Transporter protein family and does not structurally resemble Acyl-CoA synthetases. Although ALDP is clearly required for normal degradation of VLCFA, it does not have VLCS activity, and neither its biochemical function nor its role in ALD have been elucidated. However, mutations in this protein lead to devastating clinical pathology. To develop clinical therapies and to study the pathophysiology of this disease, both genetic and biochemical studies of ALDP function will be used. The VLCS gene will be cloned and its gene product characterized in order to elucidate the relationship of VLCS, previously thought to be the defect in ALD, to ALDP. Two types of mutagenesis strategies, random mutagenesis and disruption of specific regions of the regions of the protein, will be used to analyze the structure and function of ALDP. Because ABC proteins often require interaction with other proteins for function, genetic and biochemical approaches will be used to elucidate ALDP-protein interactions. Finally, the effect of ALDP on VLCFA metabolism in several cell types (including cells of neural origin) and in model membranes will examined. These studies will lead to a better understanding of ALDP function and the precise roles of ALDP and VLCFA in the pathogenesis of ALD and perhaps provide clues for the phenotypic variability characteristic of ALD.